6
C. ELSNER ET AL.
Bordusa F. 2002. Proteases in organic synthesis. Chem Rev
102:4817–4867.
hydrolysis rates were observed which follows the typ-
ical primary specificity profile of trypsin and chymotryp-
sin for specific/semispecific acyl donor ester substrates
which is characterized by a better acceptance of large
hydrophobic than small amino acid residues in P1
(Braun et al. 2000). Moreover, only a negligible hydroly-
sis of proline was found which is hard to distinguish
from the spontaneous hydrolysis. Thus, the concept of
S1–P1’ interactions seems not to be valid for the inves-
tigated 4-phenylacophenyl esters and it is more likely
that conventional S’–P’ interactions contribute to the
hydrolytic process. Within this context, the rate con-
stants are in the range of expectation and comparable
with other acyl donor esters which have extended S’–P’
interactions or can be considered as “activated” ester
substrates (Bordusa 2002). Despite Pro-4-HAB, the most
pronounced effect of the configuration of the acyl
donor ester leaving group has been found if the P1
position of the substrate is occupied with semispecific
amino acids such as Ala and Gly. This effect is reduced
or completely lost if the P1 position is occupied with
more specific amino acids, for instance the ratio is 1 in
the case of Phe and chymotrypsin. Thus, the impact of
the leaving group configuration rises as the impact of
the P1-amino acid falls.
€
Bouas-Laurent H, Durr H. 2001. Organic photochromism
(IUPAC Technical Report). Pure Appl Chem 73:639–665.
Braun K, Mitin YV, Salchert K, Schmidt T, Kuhl P. 2000.
Synthesis and use of new semispecific substrates for
trypsin-catalyzed peptide bond formation. Biocatal
Biotransform 18:427–441.
Ercole F, Davis TP, Evans RA. 2010. Photo-responsive systems
and biomaterials: photochromic polymers, light-triggered
self-assembly, surface modification, fluorescence modula-
tion and beyond. Polym Chem 1:37.
€
Gunther R, Elsner C, Schmidt S, Hofmann H-J, Bordusa F.
2004. On the rational design of substrate mimetics: the
function of docking approaches for the prediction of pro-
tease specificities. Org Biomol Chem 2:1442–1446.
€
Gunther R, Thust S, Hofmann H-J, Bordusa F. 2000. Trypsin-
specific acyl-4-guanidinophenyl esters for a-chymotrypsin-
catalysed reactions. Eur J Biochem 267:3496–3501.
Harper E, Berger A. 1972. On the size of the active site in
proteases: pronase. Biochem Biophys Res Commun
46:1956–1960.
Harris JL, Backes BJ, Leonetti F, Mahrus S, Ellman JA, Craik
CS. 2000. Rapid and general profiling of protease specifi-
city by using combinatorial fluorogenic substrate libraries.
Proc Natl Acad Sci USA 97:7754–7759.
Hedstrom L. 2002. Serine protease mechanism and specifi-
city. Chem Rev 102:4501–4523.
Kojima M, Nebashi S, Ogawa K, Kurita N. 2005. Effect of solv-
ent on cis-to-trans isomerization of 4-hydroxyazobenzene
aggregated through intermolecular hydrogen bonds.
J Phys Org Chem 18:994–1000.
Ma W, Tang C, Lai L. 2005. Specificity of trypsin and chymo-
trypsin: loop-motion-controlled dynamic correlation as a
determinant. Biophys J 89:1183–1193.
Mart RJ, Allemann RK. 2016. Azobenzene photocontrol of
peptides and proteins. Chem Commun (Camb) 52:
12262–12277.
Pearson D, Downard AJ, Muscroft-Taylor A, Abell AD. 2007.
Reversible photoregulation of binding of alpha-
chymotrypsin to a gold surface. J Am Chem Soc 129:
14862–14863.
Renner C, Moroder L. 2006. Azobenzene as conformational
switch in model peptides. ChemBioChem 7:868–878.
Riyad YM, Naumov S, Griebel J, Elsner C, Hermann R,
Siefermann KE, Abel B. 2014. Optical switching of azophe-
nol derivatives in solution and in polymer thin films: the
role of chemical substitution and environment. Am J Nano
Res Appl 2:39–52.
Conclusion
In summary, we have examined a light-induced sub-
strate activation concept for serine proteases based on
the photo-responsive properties of azobenzene as a
component of the acyl donor ester leaving group. It
was demonstrated that photo-induced leaving group
isomerization of acyl donor ester substrates of the
hydrolases trypsin and chymotrypsin has an effect on
the enzymatic reaction rate, and a discrimination of
the cis/trans reaction rate by one order of magnitude
was achieved in the best cases. Further improvement
of the concept which may be applicable to other
hydrolases needs an extended substrate leaving group
design concerning more enhanced but configuration-
discriminating S’–P’ interactions and solubility in aque-
ous systems.
€
ꢀ
Steinwand S, Halbritter T, Rastadter D, Ortiz-Sanchez JM,
Burghardt I, Heckel A, Wachtveitl J. 2015. Ultrafast spec-
troscopy of hydroxy-substituted azobenzenes in water.
Chemistry 21:15720–15731.
Disclosure statement
Thormann M, Thust S, Hofmann HJ, Bordusa F. 1999.
Protease-catalyzed hydrolysis of substrate mimetics
(inverse substrates): a new approach reveals a new mech-
anism. Biochemistry 38:6056–6062.
No potential conflict of interest was reported by the authors.
Westmark PR, Kelly JP, Smith BD. 1993. Photoregulation of
enzyme activity. Photochromic, transition-state-analog
inhibitors of cysteine and serine proteases. J Am Chem
Soc 115:3416–3419.
References
Bandara HMD, Burdette SC. 2012. Photoisomerization in dif-
ferent classes of azobenzene. Chem Soc Rev 41:180925.